Robust high content assessment of heterogeneous drug effects in 3D microenvironments

Compared to time consuming and costly in vivo experiments on drug effects at single cell level, in vitro conditions offer increased throughput for evaluating molecular interventions in a controlled context. However, our growing appreciation for the role of the extracellular microenvironment in cellular homeostasis calls into question whether typical cell culture conditions can reflect the outcome of interventions in vivo. As a result in vitro assays have been developed to incorporate more complex microenvironmental parameters, but at the cost of increasing experimental complexity. Optimization of measurement and experimental parameters is necessary to maximize the throughput and precision of these more complex experiments. Here we develop a methodology to quantify proliferation and viability of single cells in 3D culture conditions, leveraging automated microscopy and image analysis to facilitate reliable and high-throughput measurements. The single cell resolution of this approach enables stratification of heterogeneous populations of cells into differentially responsive subtypes, and we detail experimental conditions that can be adjusted to increase either throughput or robustness of the assay. Specifically we evaluate the effect of treatment time, sample volume, and cell fate detection methods, including flow cytometry, wide field fluorescence microscopy, and light sheet fluorescence microscopy. Applying this approach to a test case, we evaluate the effectiveness of a combination of RAF and MEK inhibitors on melanoma cells, we show that cells cultured in 3D collagen-based matrices are more sensitive than cells grown in 2D culture, and that cell proliferation is much more sensitive than cell viability. Surprisingly, we find that cells grown in 3D cultured spheroids exhibit equivalent sensitivity to single cells grown in 3D collagen, suggesting that for the case of melanoma, a 3D single cell model may be equally effective as 3D spheroids models. We also illustrate the importance of measuring cell fates at the single cell by showing heterogeneity in cell responses to drug treatment. Furthermore, we show that spheroids grown from single cells exhibit dramatic heterogeneity in drug response, suggesting a heritable drug resistance can arise stochastically in single cells but be retained by subsequent generations. In summary, image-based analysis renders cell fate detection robust, sensitive, and high-throughput, enabling cell fate evaluation of single cells in more complex and realistic microenvironmental conditions.